Name: Conditional Rod Cell Ablation in Xenopus Tadpoles Using the NTR-MTZ System
Uploaded: 2023-10-13T12:00:00
Description: Watch this Scientific Journal Video about Conditional Rod Cell Ablation in Xenopus Tadpoles Using the NTR-MTZ System at JoVE.com

conditional rod cell ablation in xenopus tadpoles using the ntr-mtz system

Xenopusの細胞修復戦略を調査するための網膜変性の生成

Millions of people worldwide are afflicted with various retinal degenerative diseases leading to blindness, such as retinitis pigmentosa, diabetic retinopathy, or age-related macular degeneration (AMD). To date, these conditions remain largely untreatable. Current therapeutic approaches under evaluation include gene therapy, cell or tissue transplantations, neuroprotective treatments, optogenetics, and prosthetic devices. Another emerging strategy is based on self-regeneration through the activation of endogenous cells with stem cell potential. M&#252;ller glial cells, the major glial cell type of the retina, are among cellular sources of interest in this context. Upon injury, they can dedifferentiate, proliferate, and generate neurons1,2,3. Although this process is very effective in zebrafish or Xenopus, it is largely inefficient in mammals.
Nonetheless, it has been shown that appropriate treatments with mitogenic proteins or overexpression of various factors can induce mammalian M&#252;ller glia cell-cycle re-entry and, in some cases, trigger their subsequent&#160;neurogenesis commitment1,2,3,4,5. This remains, however, largely insufficient for treatments. Hence, increasing our knowledge of the molecular mechanisms underlying regeneration is necessary to identify molecules able to efficiently turn M&#252;ller stem-like cell properties into new cellular therapeutic strategies.
With this aim, we developed several injury paradigms in Xenopus that trigger retinal cell degeneration. Here, we present (1) a mechanical retinal injury that is not cell type-specific, (2) a conditional and reversible cell ablation model using the NTR-MTZ system that targets rod cells, (3) a CRISPR/Cas9-mediated rhodopsin knockout, a model of retinitis pigmentosa that triggers progressive rod cell degeneration, and (4) a CoCl2-induced cytotoxic model that according to the dose can specifically target cones or lead to broader retinal cell degeneration. We highlight the particularities, advantages, and disadvantages of each paradigm.

著者スポットライト:Xenopus Frogの網膜再生メカニズムの探索 

Xenopusオタマジャクシにおける網膜損傷モデルの生成

My lab works on retinal regeneration. We are using the Xenopus frog as a model system. This amphibian is indeed quite fascinating because unlike mammals, like us, it can regenerate its retina very efficiently in case of injury.And we are studying the underlying mechanism because, in the future, it could be useful to trigger retinal regeneration in human patients afflicted with retinal neurodegenerative diseases. We have recently discovered that in addition to stem cells in the periphery of the retina and the retinal pigmented epithelium, neuroglial cells can also be recruited for retinal regeneration in case of injury. And so we are now studying the links between neuroinflammation and the regenerative capacity of these cells.Indeed, it seems that the neuroinflammatory niche is a key player in the modulation of the regeneration of the retina. To study the cellular and molecular mechanisms involved in retinal regeneration, we developed several retinal injury paradigms in Xenopus. The first is a mechanical retinal injury.The second is a transgenic line allowing for nitroreductase-mediated photoreceptor conditional ablation. The third is a retinitis pigmentosa model based on CRISPR/Cas9-mediated rhodopsin knockout, and, to finish, a cytotoxic model driven by intraocular injection of cobalt chloride or CoCl2. My host laboratory has showed that although Xenopus can regenerate its retina, the efficiency is highly variable and depends on the stages of the tadpole or on species, Xenopus laevis or tropicalis.This makes Xenopus a fantastic model for illustrating the molecular mechanisms that trigger or limit retinal regeneration.

Watch this Scientific Journal Video about Conditional Rod Cell Ablation in Xenopus Tadpoles Using the NTR-MTZ System at JoVE.com

Conditional Rod Cell Ablation in Xenopus Tadpoles Using the NTR-MTZ System  

NTR-MTZシステムを用いたツ コツメガ ネオタマジャクシの条件付き桿体細胞アブレーション

まず、桿体光受容体特異的プロモーターの下でGFPおよびニトロレダクターゼ(NTR)を発現するトランスジェニック株を使用します。NTRは、プロドラッグメトロニダゾール(MTZ)を細胞毒性剤に変換し、NTR発現桿体を選択的にアブレーションします。GFPモニタリングでは、スプーンを使って麻酔をかけたオタマジャクシをそっとキャッチし、小さなペトリ皿内の湿った組織に慎重に置きます。GFP蛍光を蛍光実体顕微鏡で観察し、眼の写真画像を撮影します。オタマジャクシの入ったペトリ皿を、完全に目覚めるまで飼育水を入れた大きなタンクに慎重に浸します。網膜変性症を個別にモニタリングするには、各オタマジャクシを30ミリリットルの10ミリモルMTZ、または対照溶液中のトランスジェニック兄弟を含む小さな容器に入れます。バッチ処理の場合は、トランスジェニックオタマジャクシを1リットルのMTZ(対照溶液)を入れた大きな容器に入れます。オタマジャクシを暗闇で摂氏20度で1週間飼育し、MTZの劣化を防ぎます。7日後、蛍光実体顕微鏡でGFPを観察し、眼の写真画像を撮影します。蛍光強度の低下、または蛍光の事実上の不在でさえ、桿体の劣化を示します。MTZで治療したトランスジェニックオタマジャクシの眼は、対照と比較してGFP蛍光の減少を示し、GFP免疫染色によって網膜切片でさらに確認されました。

Research

JoVE Journal

Developmental Biology

Conditional Rod Cell Ablation in Xenopus Tadpoles Using the NTR-MTZ System

Generating Retinal Injury Models in Xenopus Tadpoles

Retinal neurodegenerative diseases are the leading causes of blindness. Among numerous therapeutic strategies being explored, stimulating self-repair ...

発生生物学

Mechanical Retinal Puncture in Xenopus Tadpoles to Study Retinal Regeneration Mechanisms

Mechanical Retinal Puncture in Xenopus Tadpoles to Study Retinal Regeneration Mechanisms  

Xenopusオタマジャクシの網膜再生機構を研究するための機械的網膜穿刺

Rod Cell Degeneration in Xenopus Tadpoles by CRISPR/Cas9-Mediated Rhodopsin Knockout

Rod Cell Degeneration in Xenopus Tadpoles by CRISPR/Cas9-Mediated Rhodopsin Knockout  

CRISPR/Cas9を介したロドプシンノックアウトによるツコノプスオタマジャクシの桿体細胞変性

Retinal Cell Degeneration in Xenopus Tadpoles by Cytotoxic Intraocular CoCl2 Injections

Retinal Cell Degeneration in Xenopus Tadpoles by Cytotoxic Intraocular CoCl2 Injections  

細胞傷害性眼内CoCl2注射によるXenopusオタマジャクシの網膜細胞変性